Flaviviruses including dengue, West Nile, and Zika viruses, pose significant threats as emerging diseases and potential bioterror agents. Despite the considerable impact of flavivirus infection on world-wide health, no antiviral therapies are available, and existing flavivirus vaccines are of limited utility. Our long-term goal is to obtain detailed structural and biochemical information regarding the flavivirus replication complex and to use this information for the development of antiviral therapeutics and vaccines. The flavivirus replication complex, consisting of virally-encoded non-structural proteins (NS), unidentified cellular proteins, and the viral RNA genome, is responsible for copying the viral genome and 5' RNA capping. However, how the various components of the replication machinery are arranged as a functional complex is not known. Specifically, the 5' RNA capping machinery is functionally and physically linked to the replication machinery as multi-domain (i.e., methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) within NS5) and larger multi- protein (i.e., NS3 and NS5) complexes, but little is known regarding how RNA synthesis and RNA capping are coordinated. We have recently determined the crystal structure of the full-length dengue virus (DENV) NS5, in which MTase and RdRp could coordinate their respective activities in both monomer and dimer configurations. In the current application, we will determine how NS5 interacts with viral RNA and the NS3 protein, two other essential components in the viral replication complex. In Aim 1, we will probe how NS5 interacts with the 5' end of viral RNA, called stem loop A (SLA), to promote viral RNA synthesis. We will determine the structure of the SLA by cryo-electron microscopy, and its interactions with NS5 by a competition binding assay. In Aim 2, we will determine specific interactions among NS3 and NS5 domains during coordination of RNA synthesis and 5' RNA capping using infectious DENV RNA. In Aim 3, we will determine the X-ray crystal structures of NS5 and its RNA complexes, including SLA, dsRNA, and a `dual' substrate that binds simultaneously to both MTase and RdRp domains to represent different steps along the viral replication pathway. The combined structural, biochemical, and virological studies will help elucidate the mechanism for coordinated RNA synthesis and capping reactions in NS5.